Molecular Techniques in Histopathology

Exploration to probe into the depths of tissue morphology and molecular pathology will continue with histotechnology as the driving force

By M. Lamar Jones, BS, HT, HTL(ASCP)CM

Pathology for the most part concentrates on the morphological aspects of the study of disease. As an adjunct to pathology, molecular techniques are incorporated to isolate and identify specific agents in infectious disease, provide more accurate means of disease diagnosis, define the role of differential gene expression in disease etiology and provide personalized medicine approaches to therapy.

Molecular techniques have become a vital tool in both the clinical and anatomical pathology laboratories. In anatomical pathology many of the molecular applications have proven their value and success. Molecular techniques can define better disease, provide a more accurate diagnosis, identify predictive and prognostic markers and contribute to the development of novel molecular targets for particular therapeutic techniques. Some of the presently utilized molecular techniques in pathology and histopathology are analyzing RNA and DNA by in situ hybridization or fluorescent in situ hybridization (FISH) polymerase chain reaction (PCR).

Personalized Medicine
The era of personalized medicine arrived; the basis of treatment plans is now often built around the patient, the disease itself and how to manage the prognosis and therapy response such as cancer. One of the histological basis of molecular assays is immunohistochemistry (IHC). IHC has the ability to confine tissue cell proteins and project molecular assays and treatment plans to better manage the patient's cancer. The use of IHC to study cellular markers that often identify certain phenotypes can provide vital diagnostic, prognostic and predictive information important to disease biology.

IHC has been a continuing effort to improve sensitivity for the detection of unique antigenic targets with the primary goal of incorporating tissue-based testing with proteomic information. The use of antibodies to the molecular study of tissue pathology has required the adaption and improvement of IHC methodologies in fixed tissue specimens.

IHC and in situ hybridization is often used for recent diagnosed tumors. In some cases multigene expression outlines by molecular technologies have been implemented but the outcomes are often not known. IHC and molecular assays can be grouped together to compliment the testing for cancer diagnosis in areas such as colon, head and neck, prostate and breast cancers. IHC has been a forerunner in the development of GIST, Her2, CISH / FISH and microsatellite instability (MSI), to name a few. Certainly with new and improved monoclonal antibodies, the use of IHC on certain tumors has projected new molecular testing applications. In histotechnology, IHC has paved the way for molecular testing.

Laser Capture Microdissection (LCM)
The development of LCM is most helpful for isolating specific cells or cell population from tissue sections such as fixed or frozen sections to be used in other assays such as genetic analysis. One of the unique benefits of microdissection is the production of morphologically confirmed cellular populations. One histological use of microdissection is the molecular testing for "tissue floaters" to separate particles of possible floater from the remainder of the tissue sample. Another application is measurement of rare individual neoplastic cells such as the isolation of Reed-Sternberg cells from surrounding lymphoid infiltrates.

In situ Hybridization
In situ hybridization is a technique that can be utilized to detect certain DNA/RNA sequences, particularly in tissues. Probes rather than antibodies are used to detect sequences for DNA or RNA. Hybridization usually refers to the chemical reaction between the probe and the DNA/RNA to be detected. When performed on tissue sections or cells to detect the site where the DNA/RNA may be located, this is referred to as "in situ." This is a unique tool for identifying specific mRNA species within individual cells in tissue sections. It can indicate insights into physiological processes and diseases.

In situ hybridization techniques require many procedural steps be taken with precise optimization for each tissue tested and for each probe used. To preserve the target mRNA within tissue section, fixation in formalin is required. In situ hybridization can provide more information on the specific location of the mRNA in a cell of tissue. In situ has also been instrumental in detecting viral DNA in infectious diseases. Some of the antibodies (i.e., hepatitis B and C) and human papillomavirus (HPV) probes for in situ hybridization projected interest in early infectious diseases detection in tissue sections.

Forensics
One technique often used in forensics for the identification of DNA is "DNA fingerprinting." A reference sample is collected, which is a sample of the person's DNA. The preferred sample is usually a buccal smear, which reduces possible contamination. Blood, semen, saliva or other body fluids can also be used. This sample can be analyzed to establish a DNA profile, then the profile is compared to see if there is a genetic match. Often, the DNA fingerprinting is used to determine if a patient's tissue was accidentally switched or mixed up in the tissue preparation in the histology laboratory.

Future Aspirations
Histotechnology has evolved to become a highly technical and sophisticated science involving numerous techniques and applications for pathology. With the advent of IHC, histotechnology has risen to the forefront of molecular applications. Even the education levels for the histotechnologist have risen to meet the needs of present and future applications of advanced molecular techniques. Exploration to probe into the depths of tissue morphology and retrieve the secrets of composition to the molecular level of pathology will continue with histotechnology as the driving force.